Mechanistic investigation of capability of enzymatically synthesized polycysteine to cross-link proteins.

ABSTRACT:

Background

Previously, we had reported that ?-chymotrypsin-catalyzed polymerization of l-cysteine ethyl ester in a frozen buffer provided poly-l-cysteine (PLCys) in good yield, of which degree of polymerization had been determined to be 6-11. Almost all of SH groups in PLCys were in free forms. Such a multi-thiol peptide may cross-link proteins through thiol/disulfide (SH/SS) exchange reactions, considering the knowledge that other synthetic multi-thiol additives changes properties of protein materials.

Methods

This study explored the capability of PLCys to cross-link proteins using lysozyme as a model protein which has four disulfide bonds but no free SH group. The protein was incubated with PLCys at neutral pH and at below 70 °C to avoid PLCys-independent, ?-elimination-mediated cross-linkings. Protein polymerization was analyzed by SDS-PAGE and SEC. PLCys peptides involved in the protein polymer, which were released by reduction with dithiothreitol, were analyzed by RP-HPLC.

Conclusions

Addition of urea and thermal treatment at 60 °C caused PLCys-induced lysozyme polymerization. Compared with free cysteine, a higher level of PLCys was required for the polymerization probably due to its low water solubility. RP-HPLC analyses suggested that PLCys played a role in the protein polymerization as a cross-linker.

General significance

Enzymatically synthesized PLCys shows promise as a peptidic cross-linker for the production of protein polymers with novel physiochemical properties and functionalities.

SUBMITTER: Narai-Kanayama A 

PROVIDER: S-EPMC5613652 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature